Combined effects of nutrients on proliferation of stem cells

ABSTRACT

A method and composition for stimulating the proliferation and differentiation of stem cells is used to self-repair injury in mammals. A supplement is administered having an effective dose of blueberry, carnosine, catechin, green tea extract, VitaBlue, Vitamin D3 or combinations of these. For example, a therapeutic amount of two or more of the supplements may be selected having a synergistic effect, allowing a lower dose to achieve the same or greater effective protection as a higher dose of any one of the supplements.

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. PatentApplication No. 60/676,733 filed May 2, 2005 to Sanberg, et al., thedisclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Stem cells are found in many organs of the adult human including bonemarrow, peripheral blood, umbilical cord blood, spleen, tooth pulp, andbrain. These progenitor cells are being investigated for their potentialuse as transplanted tissues in the treatment of diseases such as cancer,diabetes, stroke, amyotrophic lateral sclerosis (ALS) and Parkinson'sdisease. Little effort however is being directed toward enhancing theendogenous stem cells in the adult as an avenue to promote healing. Inmany of these diseases, and in aging, stem cells and progenitors areknown to have a reduced proliferative activity. For example, neural stemcells, muscle satellite cells, and endothelial progenitors all showreduced proliferation in the aged and may play a role in pathology ofage-associated diseases (Kuhn et al., 1996; Conboy et al., 2005;Dimmeler and Vasa-Nicotera, 2003). In cardiovascular disease, forexample, there is a correlation between a reduction in peripheral bloodendothelial progenitor cells and many risk factors for cardiovasculardisease (Vasa et al., 2001; Hill et al., 2003). As many of the diseasesbeing targeted by stem cell therapies are age-associated diseases,selecting nutritional strategies that increase stem cell proliferationin the aged population seems appropriate.

Hematopoietic stem cells (HSCs) have been investigated for many yearsfor their utility in cancer treatments. Experimental investigations ofhematopoiesis and clinical approaches to correcting its deficiencieshave focused on cytokine activity. Cytokines modulate hematopoiesis bymaintaining the self-renewal of stem cells and stimulating theproliferation and maturation of committed progenitor cells required forthe continuous replacement of mature blood cells (Ogawa 1993; Socolovskyet al. 1998; Whetton and Spooncer 1998).

In vitro, various combinations of cytokines including interleukin-1(IL-1), IL-3, IL-6, stem cell factor (SCF), and erythropoietin (EPO)have been found to support the growth of multipotent progenitor cells(Henschler et al. 1994; Miller and Eaves 1997). Individually,granulocyte-colony-stimulating factor (G-CSF) and EPO are growth factorsfor committed myeloid and erythroid progenitors, respectively (Demetriand Griffin 1991). Clinically, G-CSF and EPO provide effectivetreatments for neutropenia and anemia (Adamson and Eschbach 1990;Eschbach et al. 1990) and are used to enhance peripheral bloodprogenitors as an alternative to bone marrow transplantation for cancerpatients. However, such treatments are costly, and are not withoutcertain risks.

Decreases in hematopoietic and endothelial progenitors are associatedwith aging. Decreases in certain hematopoietic progenitors has beenreported in frail aging women (Semba et al., 2005). Endothelialprogenitor cells (EPC) are also derived from bone marrow and found inthe circulating blood. Circulating EPC's home to sites ofneovascularization and injury (Penn et al., 2004) and can thendifferentiate into mature endothelial cells (Asahara et al., 1999).Declines in EPC's are noted in patients with coronary artery disease(Vasa et al., 2001), and when isolated from patients with high riskfactors for coronary artery disease, show increased senescence in vitro(Hill et al., 2003). It has been suggested that endothelial progenitorsplay a role in cardiovascular homeostasis and that the decline observedin aging and disease tips the balance toward injury rather than repair.Exercise has been shown to increase EPC's and this may be one of thereasons that exercise has beneficial effects on cardiovascular disease(Laufs et al., 2004). Developing nutritional based strategies toincrease progenitors could push the balance back towards repair, thushaving a significant impact on health.

Neural stem cells also decline with aging (Kuhn et al., 1996) and somehave postulated that declines in neurogenesis with aging are related tocognitive decline while others disagree (Bizon et al., 2004; Drapeau etal., 2003; Prickaerts et al., 2004). Nonetheless, it has been shown thatnutritional treatments, such as feeding with blueberry, which improvecognitive function (Joseph et al., 1999) also increase neurogenesis(Casadesus et al., 2004). Thus, there is a correlation between improvedneural stem cell proliferation and improved cognitive function.

While potentially better treatments are currently in development, fewresearch studies have investigated the effects of natural products,vitamins, and other nutrients which may modulate self-renewal of stemcells. However, in recent years there has been an upsurge of interest onthe effects of various dietary insufficiencies on hematopoietic andimmune responsiveness. Folate, vitamin 1312, and iron have crucial rolesin erythropoiesis. Erythroblasts require folate and vitamin B12 forproliferation during their differentiation. Deficiency of folate orvitamin B12 inhibits purine and thymidylate syntheses, impairs DNAsynthesis, and causes erythroblast apoptosis, resulting in anemia fromineffective erythropoiesis (Koury and Ponka, 2004). Other studies haverecently found that dietary fatty acids, particularly oleic acid andlinolenic acid, actively promote the proliferation of hematopoietic stemcells (Hisha et al., 1997; Hisha et al., 2002) as well as modulate theself-renewal of intestinal epithelial cells (Holehouse et al., 1998).Vitamin D has also received increasing attention over the past fewyears, in part, because recent studies suggest that nearly half the USpopulation may be vitamin D deficient (Meyer, 2004). Recent laboratorystudies demonstrate that vitamin D3 has a dramatic effect on stimulatingthe proliferation of various forms of multipotent progenitor cells,particularly those involved with the immune system (Mathieu et al.,2004). Recent laboratory research on cellular senescence (the end of thelife cycle of dividing cells) suggests that the dietary nutrient,carnosine, found in muscle and brain of mammals, has the remarkableability to rejuvenate cells approaching senescence, restoring normalappearance and extending cellular life span (Hipkiss et al., 1998;Holliday and McFarland, 2000).

The use of fruits or vegetables has the benefit of providing a cocktailof many different phytochemicals with multiple actions includingantioxidant and antiinflammatory effects and is one reason they havebeen extensively studied in the field of cancer biology. Other studiessuggest dietary supplementation with foods high in antioxidants, such,as blueberries, can prevent and even reverse cellular and behavioralparameters that decline as a function of aging (Joseph et al., 1999;Gemma et al., 2002). For example, dietary supplementation with 2%blueberry extract has produced both neuroprotective and neurorestorativeeffects in aged animals, perhaps as a result of modulation of cellsignaling cascades (Williams, Spencer et al. 2004). Furthermore,blueberry extract has been shown to increase neurogenesis in the agedrat brain (Casadesus, NSci Abstract, 2002). We have shown that feedingblueberries to aged rats increases the survival and growth ofhippocampal grafts grown in the anterior chamber of the eye (Willis etal., 2005), demonstrating that nutritional supplementation can not onlyincrease proliferation of tissues, but promote appropriatedifferentiation.

Green tea is a drink made from the steamed and dried leaves of theCamellia sinensis plant, a shrub native to Asia. Green tea has beenwidely consumed in Japan, China, and other Asian nations to promote goodhealth for at least 3,000 years. Recently, scientists have begun tostudy it's health effects in animal, laboratory, and observational humanstudies. Although active compounds within green tea extract have beenshown to inhibit the growth of a number of tumor cell lines, they do noteffect the growth of normal cells at similar concentrations (Chen etal., 1998; Wang and Bachrach, 2002) and actually may provide cellularprotection from aging (Song et al., 2002).

In light of such findings reviewed above, it appears that certainnutrients, vitamins, and flavonoids could have important roles inmaintaining the self-renewal of stem cells and stimulating theproliferation and differentiation of committed progenitors required forthe continuous replacement of mature cells in the blood, brain, andother tissues. Furthermore, it may be possible to use certain naturalproducts, either alone or synergistically, for the treatment ofconditions where the stem cell replacement appears warranted such asaging or diseases associated with aging. However, the amounts of suchsubstances that have shown actual results in studies are impractical toimplement as supplementation to an ordinary diet. Even if studies arecorrect about the value of these substances, consumption of sufficientquantities to substantially improve health is impractical.

Aged mammals, such as rats, dogs and humans, can improve age-relateddeclines in motor abnormalities and cognitive abnormalities with dietaryinterventions that include foods with a high antioxidant capacity.Antioxidants work at the cellular level; therefore, it would be expectedthat benefits of antioxidants in one mammal would be mirrored in othermammals. Certain foods were identified on the basis of the ability toshow antioxidant activity in vivo in mammals and in an in vitro assay.Hundreds of foods were examined using this assay (Cao et al., 1997) andseveral were chosen with high in vitro antioxidant activity for testingin vivo. For example, when 18 month old rats are fed a diet in which 2%of the diet is a blueberry extract, after 2 months on this diet, weobserve a significant improvement in motor performance on a balance beam(Joseph et al., 1999). We also observed a significant improvement on aMorris water maze in rats fed a diet supplemented with large quantitiesof strawberry, blueberry or spinach (Joseph et al., 1999). These sameanimals also show improved dopamine release in the striatum. A spinachdiet improves age-induced deficits in motor learning using either a rodrunning motor learning task or classical eye blink conditioning(Bickford et al., 2000; Cartford et al., 2002). Markers of inflammation,such as the pro-inflammatory cytokine TNFα are increased in the brainsof PD patients (Mogi et al., 1996), and 30 days following 6-OHDAlesions. We have shown that these diets decrease markers of oxidativedamage and pro-inflammatory cytokines (Gemma et al., 2002; Cartford etal., 2002), furthermore these changes are related to the foodsantioxidant activity as foods such as cucumber which are low inantioxidant activity have no effect (Gemma et al., 2002). We have beenexamining these diets in an animal model of Parkinson's disease. We havepreliminary data showing that the blueberry or spirulina diet willincrease the immune response 7 days following an insult and then preventthe prolonged activation of microglia at later time points. It is thislater prolonged activation which we hypothesize is detrimental andreflects the ongoing inflammation observed in Parkinson's disease.

While benefits are known for incorporating antioxidants into the diet ofhumans, adjusting diets to incorporate a large proportion of these foodsis difficult and often fails to incorporate sufficient amounts ofantioxidants to make a significant difference on proliferation anddifferentiation of bone marrow cells, CD34⁺ HSCs, CD133⁺ progenitorcells from peripheral blood or any other stem cells. It would be ofgreat benefit to identify certain natural compounds that can promoteproliferation of hematopoietic stem cells or other stem cells,synergistically, such that the natural compounds could be taken in theform of a supplement that would have a significant, measurable effect.

BRIEF SUMMARY OF THE INVENTION

We have discovered that certain natural products, when combined, exert asynergistic proliferation of human bone marrow cells, CD34+, and CD133+progenitors. A method of increasing stem cell proliferation and, in somecases, selective migration, and compositions have been found thatsynergistically increase the proliferation compared to individual foods,such that a combination of these natural substances shows a substantialeffect with administration of small quantities of specific naturalsubstances in mammals. Selecting at least three substances from thegroup of substances consisting of blueberry, camosine, catechin, greentea extract, and vitamin D₃, compounding the substances into asupplement easily digested in the digestive tract, and administering thesubstances to a mammal is shown to synergistically increase measurableindicators of proliferation of certain stem cells, protection againstdamage and/or damage repair mechanisms.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows data from a test of natural substances.

FIG. 2 shows a synergistic effect of a combination of naturalingredients.

FIG. 3 shows the effect of green tea in differentiation of stem cells.

FIG. 4 shows the effects on certain organs of a mammal of a process forstem cell proliferation and differentiation in mammals.

FIG. 5-7 show additional tests of stem cell proliferation anddifferentiation (FIG. 5) and protection against oxidative damage (FIGS.6 and 7).

FIGS. 8, 9 a-c show behavioral and histologic results for a test of oneexample of supplementation in mammals receiving supplementation versus acontrol group of animals.

FIGS. 10A-F, 11, 12A,B, 13A-F, 14A,B, 15A-F show test results forinduced stroke tests.

The patent or application contains at least one drawing executed incolor. Copies of this patent or patent application with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee.

DETAILED DISCLOSURE

FIG. 1 shows the results of nutrition related substances, such asextracts and compounds, promote cell proliferation of human bone marrowcells in a dose-dependent manner. Human bone marrow cells were culturedin 96-well tissue-culture plates (5×10⁴/well) and treated with humangranulocyte colony-stimulating factor (hGM-CSF; A, as positive),blueberry extract (B), catechin (C), carnosine (D), green tea extract(E) and vitamin D3 (F) at a wild range of doses as indicated for 72hours. After the treatment, these cells were prepared for MTT analysisof cell proliferation described in Materials and Methods. Data wererepresented as the percentage over control (without any treatment underthe same cultured condition). For A-F, ANOVA and post hoc testing showssignificant differences of mean percentage over control (+/−SD with n=3independent experiments) between high and low doses (p<0.005).

Three antioxidants were compared for protective effects against strokein rats: blueberry, spinach and spirulina. Results show that each, insufficient quantities in a diet, have a significant, differential effecton reducing ischemia-induced caspase-3 activity and cerebral infarction.Animals were put on a diet of either control, blueberry (10,000mg/kg/day), spinach (10,000 mg/kg/day) or spirulina (1500 mg/kg/day) for4 weeks prior to the insult. We used a 60 minute occlusion of the middlecerebral artery and at 24 hours examined the size of the infarct usingTTC staining. We found a 70% protection in infarct size in the spirulinatreated rats and a 50% protection in both the blueberry and spinachtreated rats. In these animals we have observed a significant decreasein caspase-3 activity and the number of TUNEL positive cells indicatingthat a reduction of apoptosis was achieved. All groups also showedsignificant improvement on horizontal and vertical activity measureswhen compared with controls. However, the amount of antioxidant consumedper day makes it difficult to sustain the benefits of any of these dietplans long terms.

FIG. 2 shows a Blueberry extract synergistically affects cellproliferation in the presence of co-treatment with D3, CH, D3/GT orD3/GT/Ca. In (A) Human bone marrow cells were cultured in 96-welltissue-culture plates (5×10⁴/well) and treated with blueberry extract(500 ng/mL) in the presence of D3 (5 μM), CH (20 μM), Ca (20 4M), GT(500 ng/mL), D3 (5 μM)/GT (500 ng/mL) or D3 (5 μM)/GT (500 ng/mL/Ca (20μM) for 72 hours. In (B) Human bone marrow-derived CD34+ cells(5×10⁴/well)was treated as same above (A). For MTT assay, these cellswere prepared for cell proliferation analysis. Data were alsorepresented as the percentage over control. ANOVA and post hoc testingshows significant differences of mean percentage over control (+/−SDwith n=3 independent experiment) between individual and certain combinedtreatments, for A, BB/D3 combined treatment compared to BB or D3individual treatment (p<0.005), BB/CH compared to BB or CH (p<0.005),BB/Ca compared to BB or Ca (p<0.001), BB/D3/GT compared to BB, D3 or GT,BB/D3/GT/Ca compared to BB, D3, GT or Ca; for B, BB/CH combinedtreatment compared to BB or CH individual treatment (p<0.005),BB/D3/GT/Ca compared to BB, D3, GT or Ca (p<0.001). Human CD133+ cellswere cultured for 23 days in the presence or absence of green tea (50μg/ml). Cells were then fixed and stained for A2B5 Left panels (RED),NF200 middle panels (Green) or TUJ1 right panel (red). Nuclei werestained with DAPI (Blue). In both conditions there were some cells thatwere positive for all markers which is consistent with literature thathas shown that CD133+ cells can differentiate into neural lineages. Inthe cells treated with green tea, the staining was significantly moreprevalent.

Several whole food extracts, herbal extracts, and specific compoundswere screened individually for proliferative activity on human bonemarrow cells in culture. Spinach, spirulina, EGCG, epicatechin,withania, somnifera, carao, rehmania glutinosa, and astragulusmembranaceous did not show high activity on proliferation of human bonemarrow cells in culture and were not tested further.

Certain whole-food extracts, such as blueberry, green tea, and specificcompounds, including catechin, carnosine, and vitamin D3, were found toincrease cell proliferation of human bone marrow cells in adose-dependent manner (FIG. 1 B-F). Cell proliferation, as determined byMTT assay, is displayed as the percent of cell proliferation over thecontrol, which represents cells cultured in the same condition withoutany extract or compound added.

The positive control, human granulocyte colony-stimulating factor(hGM-CSF; FIG. 1A), produced a 44.5±8.1% proliferation at the highestdose of 100 ng/ml. Blueberry and catechin demonstrated a 34.5±6.7 and34.8±5.2% increase in proliferation at 500 ng/ml and 20±M, respectively(FIG. 1 B,C). Carnosine displayed a 26.6±6.0% increase at 20±M (FIG. 1D), and vitamin D3 displayed a lower percentage of proliferation,14.8±3.3% at 5±M (FIG. 1 F). Green tea produced a proliferation similarto blueberry and catechin with 35.6±9.2% proliferation at 500 ng/ml(FIG. 1 E).

Human bone marrow cells were cultured with different combinations of theextracts and compounds and with the individual extracts and compounds atthe highest doses determined to promote the greatest amount ofproliferation, which was represented by FIG. 1 A-F. The positivecontrol, hGM-CSF displayed 48.3±7.4% proliferation. As shown in FIG. 2Ablueberry, catechin, carnosine, green tea, and vitamin D3, alone, didnot cause proliferation in a significantly different manner from thatshown in FIG. 1 A-F. However, the combination of extracts and compoundsresulted in a greater percentage of proliferation than observed with theindividual extracts and compounds. For example, blueberry/vitamin D3exhibited a 62% increase in proliferation, blueberry/catechin a 70%increase, and blueberry/carnosine with the greatest synergistic affectof 83% (FIG. 2 A). Blueberry/green tea, blueberry/vitamin D3/green tea,and blueberry/vitamin D3/green tea/carnosine also displayed significantincreases in proliferation of 56%, 72%, and 70%, respectively, in FIG.2A and significant synergistic effects are shown in FIGS. 2 B and 2 C,also.

FIG. 4 shows results for bone marrow (BM), spleen (SPL) and themononuclear fraction of peripheral blood (MNC) isolated from 3 micetreated with either water by gavage (control), a composition ofsynergistic natural substances by gavage (Composition 1 supplementation)or G-CSF i.p. (G-CSF) for 6 days. On day 7 cells were isolated andprepared for flow cytometry. The percentage of CD117/SCA-1 positivecells was analyzed. This represents a hematopoietic stem cells. As canbe seen above, there was a trend for an increase in this population ofcells in the mice given Composition 1 supplementation compared to allcell populations examined. In the G-CSF treated mice there was a trendfor an increase in the spleen and MNC populations, but a decrease in theBM population which is consistent with what is observed for otherprogenitors following G-CSF assessed by the colony forming assay. FIG. 5shows results for tests of bone marrow (BM), spleen (Spl), andmononuclear cells (MNC) isolated from mice treated with either water(control), G-CSF, or Composition 1 for 6 days. On day 7 cells wereisolated and plated in the colony forming assay (see specific methods)for 5 days. ATP was detected by luciferase assay and luminescence wasconverted to ATP units with use of a standard curve. As can be seen fromthis graph GCSF has the expected effect to reduce bone marrow CFU's andincrease CFU's found in both spleen and the MNC fraction. Composition 1shows a trend towards increasing CFU's in both spleen and MNC fractionsbut does not reduce CFU's in the BM fraction. Although a statisticallysmall sample, the effects show a cytokine like effect to reduceprogenitors in the bone marrow.

Reagents. All compounds were added to cell cultures as described in theresults sections. Sources of compounds were as follows: blueberry(freeze dried powder, Van Drunen Farms, Momence, Ill.), green teaextract (Rexall), Carnosine (Sigma), Catechin (Sigma), and the activatedform of vitamin D3 (25-Hydroxycholecalciferol, Sigma).

Cell Cultures and MTT Assay. For cell proliferation analysis, human bonemarrow cells, human CD34⁺ cells or CD133 cells (BioWhittaker, Inc.) werecultured in 96 well plates (5×10⁴/well) containing 100 μL of completemedium (RPMI 1640 medium supplemented with 5% fetal calf serum). Thesecells were treated for 72 hours with various extracts at a wide range ofdoses (8 ng/mL to 500 ng/mL) or molecular compounds (0.3125 μM to 20μM). Five hours before the end of the treatment, 20 μL of MTT solution(MTT kit, Sigma) was added to each well. These plates were thenincubated in a CO₂ incubator at 37° C. for 5 hours and the culturedmedia removed with needle and syringe. 200 μL of DMSO was added to eachwell with pipetting up and down to dissolve crystals. These plates wereput back into the 37° C. incubator for 5 minutes, transferred to platereader and measured absorbance at 550 nM. Data were represented asrelative percentage mean proliferation, defined as O.D. reading numberof each treated cells normalized to control cells (in the absence oftreatment).

Promotion of Bone Marrow Cell Proliferation in a Dose-dependent Manner.Certain whole food extracts, such as Blueberry (BB), Green Tea (GT), andspecific compounds, including Catechin (CH), Carnosine (Ca), and VitaminD3 (D3), were found to increase cell proliferation of human bone marrowcells in a dose dependent manner (FIG. 1). Cell proliferation asdetermined by MTT assay is displayed as the percent of cellproliferation over the control, which represents cells cultured in thesame condition without any extract or compound added. The positivecontrol, human granulocyte colony-stimulating factor (hGM-CSF; FIG. 1A),produced a 44.5±8.1% proliferation at the highest dose of 100 ng/mL.Blueberry (BB) and CH demonstrated a 34.5±6.7 and 34.8±5.2% increase inproliferation at 500 ng/mL and 20 μM respectively (FIG. 1B, 1C). Thecompound, Ca displayed a 26.6±6.0 increase at 20 μM (FIG. 1D), and D3displayed a lower percentage of proliferation, 14.8±3.3% at 5 μM (FIG. 1F). Green tea (GT) produced a proliferation similar to BB and CH with35.6±9.2% proliferation at 500 ng/mL (FIG. 1 E).

Synergistic Stimulatory Effect of Extracts and Compounds onProliferation. To determine if the extracts and compounds displayedsynergistic effect on cell proliferation, we cultured human bone marrowcells with different combinations of the extracts and compounds. We alsocultured the bone marrow cells with the individual extracts andcompounds at the highest doses determined to promote the greatest amountof proliferation, which was represented by FIG. 1A-F. The positivecontrol, hGM-CSF displayed 48.3±7.4% proliferation, while BB, CH, Ca,GT, and D3 alone did not cause proliferation in a significantlydifferent manner as demonstrated in FIG. 2A. However, the combination ofextracts and compounds resulted in a greater percentage of proliferationthan observed with the individual extracts and compounds. For example,BB/D3 exhibited a 62% increase in proliferation, BB/CH a 70% increase,and BB/Ca with the greatest synergistic affect of 83% (FIG. 2A). BB/GT,BB/D3/GT, and BB/D3/GT/Ca also displayed significant increases inproliferation of 56%, 72%, and 70% respectively (FIG. 2A).

Promotion of CD34⁺ Cell Proliferation and Synergistic Properties ofExtracts and Compounds. To determine whether these extracts andcompounds promoted cell proliferation of other progenitor cells, wecultured CD34⁺ human hematopoietic stem cells under the same conditionsas the bone marrow cells using different combinations of the extractsand compounds, and with the individual extracts and compounds at thehighest doses determined to promote the greatest amount of proliferationin the bone marrow cell studies, which was represented by FIG. 1A-F. Theresults revealed a 48.3±7.4 increase for hGM-CSF, which wasapproximately a 5% increase in proliferation as compared to hGM-CSFeffect on the bone marrow cells (FIG. 2B). However, individually, BB,CH, Ca, GT, and D3 displayed a 20.9±3.0, 24.8±5.0, 11.05±2.1, 14.0±3.7and 6.9±2.6 increase in proliferation respectively, which are much lowerthan observed in the bone marrow cells (FIG. 2B). However when combined,BB/D3, BB/CH, BB/Ca, BB/GT, and BB/D3/GT demonstrated a 39.3±2.0%,57.3±10.4%, 30.9±3.4%, 27.9±10.0%, and 49.9±13.1% increase inproliferation respectively which is at least additive and in somesynergistic (FIG. 2B). Interestingly, the combination of BB/D3/GT/Caresulted in an increase of 67.6±11.9%, a simple additive effect wouldhave been 52% demonstrating a synergistic (FIG. 2B).

Promotion of CD133+ Cell Proliferation and Synergistic Properties ofExtracts and Compounds. Some of the compounds and combinations with thegreatest activity on proliferation of the bone marrow derived CD34+cells were then used to treat CD133+ (progenitor cells) collected fromperipheral blood and cultured under the same conditions as above. Cellproliferation was determined by MTT Assay and is displayed as thepercent of cell proliferation over the control. The results revealed an21.11±2.9% increase after treatment with hGM-CSF (FIG. 2C).Individually, BB, Ca, GT, and D3 displayed a 11.9±3.1, 16.9±3.3,13.57±3.0, 7.6±1.39% increase in proliferation respectively (FIG. 2C).When combined, BB/D3/GT, and BB/D3/GT/Ca demonstrated a 29.2±3.6 and42.5±5.9% increases in proliferation (FIG. 2C) of human CD133⁺ cells.

In vivo studies. We have initiated studies with Composition 1administered by gavage to DBA mice. To date we have treated 3 mice with(Blueberry Extract: 3 mg/kg/day; Carnosine: 10 mg/kg/day; Vitamin D3: 1mg/kg/day; green tea extract: 3 mg/kg/day) or water by gavage, inaddition 3 mice have been treated with G-CSF 4 μg/mouse i.p.. Initialstudies were done at 6 days of treatment as this is an optimal time forG-CSF to show mobilization of progenitors into the peripheral blood.Bone marrow, spleen and peripheral blood mononuclear cells were examinedby flow cytometry with SCA-1 and CD117, the presence of both markers isindicative of multipotent hematopoietic stem cells. As can be seen inFIG. 4, there was in increase in CD117/SCA-1 double positive events inall 3 cell fractions examined in mice treated with Composition 1. Asmall increase was also observed following G-CSF. The three fractionswere also assessed in the HALO™ colony forming assay. As can be seen inFIG. 5, G-CSF produced a decrease in colony forming units—total count(CFU-C's) in the bone marrow and an increase in CFU-C's in both spleenand mononuclear cells. This effect is consistent with the literature andthus increases our confidence in this version of the CFU-C assay. Ourproduct at this dose and time of delivery showed a trend for an increasein CFU-C's in the spleen and MNC fraction. G-CSF is a potent cytokineused to increase progenitors for a short term effect; howeveradministering G-CSF beyond a short term effect causes an ultimatedecrease of bone marrow progenitors Our product is designed for longerterm administration and does not show a long term decrease in bonemarrow progenitors.

The results demonstrate that various natural compounds and theircombinations promote the proliferation of human bone marrow, human bonemarrow derived CD34⁺, and human peripheral blood derived CD133⁺ cells.When tested individually, these compounds were most effective inpromoting proliferation of the bone marrow cells and less effective whenused to treat the progenitor populations. This finding may reflect aneffect of the individual compounds on the mature cell populations thatare also present in the bone marrow cell cultures. When the activity ofthe compounds was examined in combinations, the additive and synergisticeffects were more profound in the progenitor CD34⁺ and CD133⁺ cells.Surprisingly, some of the combinations tested resulted in proliferationwhich exceeded that produced by the positive control, hGM-CSF. Forexample, the combination of blueberry extract, green tea extract,carnosine, and vitamin D3 produced greater proliferation than thatinduced by hGM-CSF in all three cell types, with CD133⁺ cells being mostsensitive with a proliferation response twice that produced by hGM-CSF.Of all the compounds tested, blueberry extract most consistentlyproduced significant proliferation when combined with the othercompounds.

In summary, we demonstrated for the first time that certain naturalcompounds can promote proliferation of hematopoietic stem cells invitro, and more specifically that a combination of blueberry extract,green tea extract, carnosine, and vitamin D3 demonstrate synergisticactivity in these assays.

Following in vivo, extension of our promising in vitro results, it islikely that an oral formulation of blueberry extract, green tea extract,carnosine, and vitamin D3 developed as a dietary supplement could beused to promote healing naturally in various parts of the body whereprogenitor cells are in need, such as in the case with various diseasestates or with an injury.

One of the ways of testing the effectiveness of an antioxidant is toshow its effects in preventing oxidative damage, such as shown in FIGS.6 and 7. Mice (BalbC) treated with supplementation for 2 weeks by gavagewere anesthetized by using isoflurane in a bell jar. After beingproperly anesthetized both hind legs were then removed, at the pelvicjoint, from the torso and placed in a Petri dish containing 70% ethanol.All tissue was then dissected away and removed from the bone, and femurand are isolated in another dish. A 1 cc syringe filled with 1 mL ofculture media (minimum essential media) and is inserted through theendplate of the femur while the other end is removed using scissors. Thebone marrow is then washed through the bone into another dish thatcontains 10 mL of culture media. The bone is washed several times toensure sufficient marrow collection. This process is repeated for thefibia and samples from both legs are collected together and cell numberwas assessed using a hemacytometer. Cells were then cultured at 5×106cells per well and incubated at 37° C. for 2 hrs. The bone marrow waschallenged using a serial dilution of H₂O₂ ranging from 1000 μM-7.8 μM,and incubated over night at 37° C. The culture media was collected andcentrifuged at 14,000 rpm for 10 min at 4° C. Supernatants weretransferred to fresh tubes and the LDH levels were tested using theCytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) in strictaccordance with the manufacturers' protocols. Results were thencalculated to show the percent cell death for the control and treatmentgroups, using varying doses of peroxide (+/−SD) as the oxidant.

FIG. 6 shows data from a sample of 3 mice per group of BalbC micetreated with either water Composition 1. The drop in LDH release (in %)−/+SD (ordinate) is shown against the amount of oxidant used in μM(abscissa). As the amount of oxidant used increases, (from right toleft), the amount of LDH released increases faster for the control groupwith water than for the group protected by Composition 1 In FIG. 7,results are shown for mice treated as described in the tests of FIG. 6.Composition 2 and 3 (see below) were administered for 2 weeks at 2different doses. The first dose is exactly equivalent to the doseprovided in a formulation for humans, and the higher dose is 10 foldincrease, because the metabolism of a mouse is 10 times higher thanhumans. Thus the higher dose in the mouse will produce similar bloodlevels of components to the dose used in the formulation for humans, andthe blood level is thought to be the significant measure ofconcentration in comparisons between mammals.

Composition 2 for the human formulation comprised Green tea of 5.7mg/kg, Carnosine of 1.4 mg/kg, Vitamin D3 of 0.71 μg/kg, Blueberry=5.1mg/kg, and Vitablue=0.6 mg/kg. The composition for the formulationneeded to produce a similar blood level in mice was administered at 10times the human formulation (Composition 3). Significant protection isprovided at both doses compared to the control group. The humanequivalent dose shows a marked protection against oxidative damage.

An example of a supplement formulation may include 360 mg of blueberry.Alternatively, an equivalent amount of VitaBlue may be added. It isthought that anthocyanins are the active ingredient in blueberries, andVitaBlue is enriched by ten times in anthocyanins compared to blueberry.Thus, it is believed that 40 mg of VitaBlue is equivalent to 400 mg ofblueberry. A pharmacokinetic study (pK study) in humans [Mazza G, Kay CD, Cottrell T, Holub B J (2002) Absorption of Anthocyanins fromBlueberries and Serum Antioxidant Status in Human Subjects J. Agric.Food Chem., 50, 7731-7737] (7) found consumption of 1200 mg blueberryanthocyanins resulted in human plasma conc. of 17 ng/ml. The equivalentof 800 mg of blueberry (1.2% anthocyanins) would lead to a bloodconcentration of approximately 0.14 ng/ml anthocyanins by interpolation.The doses tested in vitro ranged from 0.08 to 5 ng/ml anthocyanins. Inhumans, this range corresponds to a dose in a range from 400 mg to 25grams of blueberries and/or their equivalent. The upper range has beentested as a supplement in our tests for aging and other studies. Inpractical terms, 25 grams of blueberry would be about 12 pills, which isimpractical for supplements provided in pill form. More preferably, theupper limit for blueberry and equivalents is about 5 grams per day. Evenmore preferably, blueberry is compounded with other supplements thatprovide a synergistic effect.

Our in vitro data for activity of green tea extract tested a range from4 ng/ml to 250 ng/ml catechins (assuming only 10% of the catechins wentinto solution, we obtain a range of 0.4-25 ng/ml). A pK study in humans[Manach C, Gary Williamson, Christine Morand, Augustin Scalbert, andChristian Rèmèsy (2005) Bioavailability and bioefficacy of polyphenolsin humans. I. Review of 97 bioavailability studies. Am J Clin Nutr; 81(suppl):230S-42S] (6) found that consumption of 500 mg catechinsresulted in a plasma conc of 2 nmol/l. Using the MW of catechin at 280,2 nmol/l=0.58 ng/ml. Thus, in humans 400 mg GTE will result in a 0.4ng/ml plasma concentration, and a preferred range of green tea extractis from 400 mg to 25 grams. For practical considerations, the amount ofgreen tea extract is selected to be no greater than 5 grams. Morepreferably, green tea extract is compounded with other substances toprovide a synergistic effect.

In one example, Vitamin D3 is used as 2000 IU's, which is equivalent to50 μg. In humans, daily administration of 4000 IUs (100 mcg) results ina blood concentration of 100 nmol/L. There is no official RDA forvitamin D3 [R. Vieth, D. Fraser, Vitamin D insufficiency: no recommendeddietary allowance exists for this nutrient, CMAJ 166 (2002) 1541-1542](4). According to a conservative report of the Food and Nutrition Board[R. Vieth (2004) Why the optimal requirement for Vitamin D3 is probablymuch higher than what is officially recommended for adults. Journal ofSteroid Biochemistry & Molecular Biology 89-90 (2004) 575-579], thesafety limit (no adverse effect level, NOAEL) of vitamin D3 intake inhumans is 60 mcg (or 2400 IU) per day [Standing Committee on theScientific Evaluation of Dietary Reference Intakes. Dietary ReferenceIntakes: Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride.National Academy Press, 1997]. After applying a margin of safety, therecommended upper limit is 50 mcg (2000 IUs) per day for intake by thegeneral public [I. Munro, Derivation of tolerable upper intake levels ofnutrients, Am. J. Clin. Nutr. 74 (2001) 865-867]. Clinical trials showno benefit from oral doses 20 mcg (800 IUs) or less [R. Vieth (2004) Whythe optimal requirement for Vitamin D3 is probably much higher than whatis officially recommended for adults. Journal of Steroid Biochemistry &Molecular Biology 89-90 (2004) 575-579] (1). Recent, clinical trialsalso report that human oral doses of 1000 IUs (20 mcg/day) to 4000 IUs(100 mcg/day) are completely safe and within normal levels produced byexposure to the sun (total body sun exposure=10,000 IUs/day) [R. Vieth(2004) Why the optimal requirement for Vitamin D3 is probably muchhigher than what is officially recommended for adults. Journal ofSteroid Biochemistry & Molecular Biology 89-90 (2004) 575-579] (1). Adose of 50 mcg leads to a plasma concentration of 0.05 μM. Doses testedin vitro ranged from 0.07 to 5 μM, a range of 50 μg to 5000 μg inhumans, which is from 2000 IU to 20,000 IU. In practice, 20,000 IU's isbeyond any limit recommended and might lead to hypercalcemia. In apreferred example, the range of Vitamin D3 is selected in a range from2,000 to 4,000 IU.

In one example, camosine is added to a supplement in a dose of 100 mg. Arecent pK study in humans [Park Y J, Volpe S L, Decker, E A (2005)Quantitation of Carnosine in Humans Plasma after Dietary Consumption ofBeef. J. Agric. Food Chem., 53, 4736-4739] found that two beef pattiescontain 250 mg carnosine and when ingested by humans resulted in aplasma conc. of 33 mg/l which peaked at 3 hrs and returned to baselineat 5 hrs. With a Mw of 266 and assuming 5 liters of blood in a human,250 mg carnosine produces a blood conc of 620 μM. Assuming linear pK,100 mg should produce a human blood concentration of 248 μM. Dosestested ranged from 1-20 μM, effect was still increasing at 20 μM. Thus,a dose as high as 620 may be helpful and still be within normal rangesfor human consumption. A preferred effective range is about 10 mg to 100mg of camosine based on extrapolation from results obtained.

In one preferred example, at least three substances are selected to becompounded into a supplement tablet, pill, or other form for ease ofadministering a dose. In humans, a ratio of blueberry to Vitamin D3 maybe selected from 400:0.1 to 1:4000 or 25,000:0.05 to 1:500,000, based onthe previous maximum and minimum ranges for each. However, limiting thedoses of each to 5 grams, yields ratios from 5,000:0.5 to 1:100,000 forthe highest to lowest ranges of each component.

Similarly, a ratio may be calculated for blueberry to green tea. Thus,the lowest to highest ratio is in a range from 4:50 to 0.08:1, and thehighest to lowest ratio is in a range from 50:4 to 12.5:1.

In another example, several ratios may be selected for a combination ofblueberry to carnosine to green tea extract to Vitamin D3. Exampleswould include from 4,000:4,000:100:1, such as a composition havingVitamin D3 at the recommended highest level, or 100,000:100,000:2,000:1,with Vitamin D3 at its low level.

Two groups of adult male Sprague-Dawley rats initially receivedsupplementation using Composition 1 (n=8) or vehicle (n=7). Composition1 equals (blueberry 3 mg/kg/day; Vitamin D3 1 mg/kg/day; green tea 3mg/kg/day; and carnosine 10 mg/kg/day. Dosing for Composition 1 andvehicle consisted of daily oral administration (using a gavage) over atwo-week period.

We tested a pre-stroke diet supplementation dosing regimen using dailyadministration of supplementation for two weeks. Following the lastsupplementation, on day 14, all animals underwent a stroke surgery usinga transient one-hour suture occlusion of middle cerebral artery (MCAo).This MCAo stroke model was used to test the neuro-protection ofsupplementation for some examples of the present invention. To revealthe functional effects of supplementation, animals are subjected toestablished behavioral tests just prior to stroke surgery and again onday 14 post-stroke. Behavioral tests include Bederson test and elevatedbody swing test (EBST), which are sensitive to stroke-induced motor andneurological deficits, respectively.

The Bederson test is conducted following the procedures previouslydescribed (Borlongan et al. 2004a,b). Neurologic score for each rat isobtained using 4 tests which include: (1) observation of spontaneousipsilateral circling, graded from 0 (no circling) to 3 (continuouscircling); (2) contralateral hindlimb retraction, which measures theability of the animal to replace the hindlimb after it is displacedlaterally by 2 to 3 cm, graded from 0 (immediate replacement) to 3(replacement after minutes or no replacement); (3) beam walking ability,graded 0 for a rat that readily traverses a 2.4-cm-wide, 80-cm-long beamto 3 for a rat unable to stay on the beam for 10 seconds; and (4)bilateral forepaw grasp, which measures the ability to hold onto a2-mm-diameter steel rod, graded 0 for a rat with normal forepaw graspingbehavior to 3 for a rat unable to grasp with the forepaws. The scoresfrom all 4 tests are added and the average calculated to give aneurologic deficit score (maximum possible score, 3).

The EBST involves handling the animal by its tail and recording thedirection of the swings. The animal is gently picked up at the base ofthe tail, and elevated by the tail until the animal's nose is at aheight of 2 inches (5 cm) above the surface. The direction of the swing,either left or right, is counted once the animals head moves sidewaysapproximately 10 degrees from the midline position of the body. After asingle swing, the animal is placed back in the Plexiglas box and allowedto move freely for 30 seconds prior to retesting. These steps arerepeated 20 times for each animal. Normally, intact rats display a 50%swing bias, that is, the same number of swings to the left and to theright. A 75% swing bias would indicate 15 swings in one direction and 5in the other during 20 trials. We have previously utilized the EBST, andnoted that MCAo stroke animals display >75% biased swing activity asearly as the day of stroke surgery (i.e., after recovery fromanesthesia), and such motor asymmetry is stable for up to six months(46, 50). All tests were conducted by two investigators blinded to thetreatment condition.

FIG. 8 compares results for neurologic (Bederson test) and motor (EBST)evaluations prior to stroke surgery (pre-stroke), which revealed nodetectable behavioral deficits between groups, and at 14 days afterstroke (post-stroke). Post-stroke, while both groups exhibited motor andneurologic deficits (versus pre-stroke: *p's<0.05), the rats havingComposition 1 supplementation exhibited significantly less motor andneurologic deficits than the control group (**p's<0.05). ANOVAstatistical analysis revealed significant treatment effects in bothBederson (F_(3,26)=81.65, p<0.0001) and EBST (F_(3,26)=29.26, p<0.0001),as shown by the error bars displayed in FIG. 8. Pair-wise comparisonsbetween treatment groups using Fisher's PLSD posthoc t-tests revealedComposition 1 supplementation and vehicle-treated groups displayed nodetectable behavioral impairments at pre-stroke testing; however, bothexhibited motor and neurologic deficits at post-stroke testing (comparedto pre-stroke: p's<0.05). The rats having Composition 1-supplementationshowed significantly improved motor and neurologic tests compared to thecontrol group (at post-stroke testing: p's<0.05). Reductions of 11.8%and 24.4% in EBST motor asymmetry and Bederson neurologic dysfunction,respectively, were detected with Composition 1-supplementation comparedto the control group.

Following the behavioral testing at day 14 post-stroke, all animals wereeuthanized for evaluation of cerebral infarction using the glialfibrillary acidic protein (GFAP) immuno-staining (Borlongan et al.2000). The GFAP assay is a well accepted method to reveal the extent ofany glial scar, which closely correlates with cerebral damage in strokevictims. Using an NIH imaging system, the glial scar was calculated bycapturing images using AxioPhot (Carl Zeiss) at 1.6-fold magnification.The damaged area was selected according to the morphology of the cellsbased on glial infiltration which clearly delineated the ischemic corefrom the ischemic penumbra (Borlongan et al. 2000). The mean area ofdamage of 5-6 sections per coronal slice was calculated using thefollowing formula: C=D/(A−B), to reveal the total infarct area perbrain, with areas A, B, C and D being defined as shown in FIG. 9 a, forexample.

The t-test results of FIG. 9 c show that Composition 1 supplementationreduces the glial scar/ischemic area damage in the striatum compared tothe control group (Vehicle). A representative comparison is shown in themicrographs of FIG. 9 a (Composition 2 supplementation) and FIG. 9 b(Vehicle). A significant decrease in mean glial scar area (e.g. 75%) wasobserved in the ischemic striatum of animals having Composition 1supplementation compared to that of vehicle-treated animals (p<0.0005)as shown by the error bars in FIG. 9 c. These histological resultsparallel the improved behavioral performance for Composition 1supplementation found in the Bederson Test and EBST.

The results that correlate Composition 1 supplementation to cellproliferation in vitro also occurs in vivo. It is believed, withoutbeing limiting, that stem cell proliferation serves as the mechanisticexplanation for the observed improvements in behavioral and histologicalobservations for induced stroke animals compared to the control group.Alternate brain sections obtained from the same Composition 1supplementation or control animals were processed for BrdUimmunostaining (Sigma, 50 mg/kg, i.p. every 8 hours during days 10 to 14post-stroke) to reveal cell proliferation. Analyses of BrdU labelingwere focused at the neurogenic subventricular zone (SVZ) and thenon-neurogenic striatum. Focusing on SVZ is used to reveal any effect ofComposition 1 supplementation on increased cell proliferation in aneurogenic site. An increased cell proliferation in SVZ may be used toshow support of a mechanism for neuro-protection using Composition 1supplementation. In addition, Analyzing BrdU labeling in the ischemicstriatum reveals whether newly formed cells from the SVZ are able tomigrate towards the site of an induced-stroke injury. Evidence ofmigration of newly proliferated stem cells to a site of injury providesthe strongest possible evidence for the efficacy of Composition 1supplementation in the mechanism responsible for protection frominduced-stroke damage.

The images of FIG. 10 A-F show BrdU labeling of brain tissues.Immunofluorescence microscopy was used to visualize cell proliferationin SVZ and ischemic striatum of animals given Composition 1supplementation compared to a control group. FIG. 10 D-F were taken fromanimals given Composition 1 supplementation. These micrographs arecharacterized by significantly increased BrdU labeling in both SVZ andischemic striatum of brain sites compared to the BrdU labeling of thesame brain sites of animals in the control group, which are shown inFIG. 10 A-C. Composition 1 increased the number of BrdU-positiveproliferating cells in SVZ (panel D in rectangle and magnified in E) andischemic striatum (panel D in circle and magnified in F) compared tovehicle treatment (SVZ: panel A in rectangle and magnified in B;striatum: panel A in circle and magnified in C).

In FIG. 12 quantitative analysis of SVZ's cell proliferative activity,as shown in the micrograph of FIG. 11 (protocol based on Baldauf andReymann, 2005) revealed significant treatment effects (F_(3,16)=18.03,p<0.0001), with at least a one-fold increment in the number ofBrdU-positive cells in the Composition 1 supplementation stroke brainscompared to control stroke brains (p<0.0005) (FIG. 10 A,D in rectanglesand magnified in B,E; quantitative data shown in FIG. 12 A). Similarly,quantitative analysis of BrdU labeling in the ischemic striatal penumbrarevealed significant treatment effects (F_(3,16)=11.84, p<0.0001), withat least a three-fold increase in the number of BrdU-positive cells inthe Composition 1 treated stroke brains compared to vehicle-treatedstroke brains (p<0.0001) (FIG. 10 A,D in circles and magnified in C,F;quantitative data shown in FIG. 12 B). In contrast, when evaluation ofBrdU labeling targeted the ischemic core (instead of the penumbra, seeFIG. 1 A,D), there was a massive proliferation of BrdU-positive cells inthe vehicle-treated stroke brains compared to Composition 1 treatedstroke brains. This apparent increase in BrdU labeling in thevehicle-treated ischemic core might be misunderstood as a sign ofproliferation, but may be understood as being established by BrdU labelsinfiltrating reactive microglia, as well as degenerating or dead cellswhich are abundant in the necrotic core (Borlongan et al. 2000; Ito etal. 2001; Beech et al. 2001; Marks et al. 2001). Since Composition 1treated stroke brains have smaller ischemic core, it is expected thatBrdU labeling in this region is less than that of the vehicle-treatedstroke brains. Accordingly, examination of cell proliferation in theischemic core is known to present as an artifact that might not trulyreflect a neuroprotective BrdU labeling index. With this in mind, theevaluation of cell proliferation is limited to SVZ and ischemicpenumbra, which are not known to present as an artifact. Both these setsof data indicate that Composition 1 increases cell proliferation in theneurogenic SVZ, and also facilitated the migration of these newly formedcells towards the ischemic striatal penumbra. Quantitative analysis ofBrdU labeling in SVZ followed the protocol by Baldauf and Reymann(2005). Four rectangular sections (200 um×60 um) at 100× magnificationfrom two serial brain sections from each rat were used to reveal meanBrdU cell counts along the SVZ. For quantitative analysis of BrdUlabeling in striatum, two serial brain sections from each rat capturingthe striatal penumbra depicted in FIG. 10 A,D (magnified in FIG. 5 C,E),with each section corresponding to 40 um×40 um, were used to reveal meanBrdU cell counts in the ischemic striatum. Results revealed one-fold andthree-fold increments, respectively, in the SVZ results of FIG. 12 A andstriatum results of FIG. 12 C for the Composition 1 treated strokebrains compared to vehicle-treated stroke brains (*p<0.0005 in SVZ and*p<0.0001 in striatum when comparing corresponding ischemic SVZs andstriata between treatment groups).

Composition 1 leads to increased expression of neuronal phenotypes,bolstering the evidence of a mechanism of neurogenesis underlyingComposition 1 neuro-protection. The BrdU-labeled brain sections (i.e.,ischemic striatal penumbra) used for cell migration studies above werealso labeled with the neuronal marker doublecortin or glial marker GFAP.Composition 1 enhanced neuronal differentiation of newly formed cells inthe ischemic striatum is revealed by a high number of BrdU (D) anddoublecortin (E) double-labeled cells (F) compared to vehicle treatment(A: BrdU, B: doublecortin, C: merged). Immunofluorescence microscopyrevealed widespread double-labeling of cells with BrdU and doublecortinin Composition 1 treated stroke brains (FIG. 13 D-F). In contrast, onlya few cells double-labeled with both BrdU and doublecortin invehicle-treated stroke brains (FIG. 13 A-C).

Quantitative analysis revealed significantly higher double-labeling ofBrdU and doublecortin in Composition 1 than vehicle-treated strokebrains (*p<0.05, **p<0.0001) of FIG. 14 A. In contrast, there weresignificantly lower BrdU and GFAP double-labeling in Composition 1 thanvehicle-treated stroke brains (**p's<0.0001) of FIG. 14 B. Quantitativeanalysis revealed about 17% and 75% double-labeling of BrdU anddoublecortin in respective intact and infarcted side of Composition 1treated stroke brains, which were significantly higher than those seenin the intact (5%) and infarcted side (13%) of vehicle-treated strokebrains (*p<0.05, **p<0.0001) as shown in FIG. 14 A.

In contrast, only a few cells in Composition 1 treated stroke brainsdouble-labeled with BrdU and GFAP (FIG. 15 D-F), while many cells invehicle-treated stroke brains double-labeled with BrdU and GFAP (FIG. 15A-C). The GFAP marker differentiates glial lineage. In FIG. 15 A-F,Composition 1 supplementation did not enhance differentiation of newlyformed cells into glial lineage in the ischemic striatum, as revealed bya few cells with BrdU (D) and GFAP (E) double-labeling (F), while cellsreveal double-labeling with BrdU and GFAP in the vehicle treatedischemic striatum (A: BrdU, GFAP, C: merged).

Quantitative analysis revealed about 1% and 2% double-labeling of BrdUand GFAP in respective intact and infarcted side of Composition 1treated stroke brains. The analysis of intact (18%) and infarcted (35%)sides of vehicle-treated stroke brains showed significantly higherdouble-labeling (**p's<0.0001) (FIG. 9B). These data show thatComposition 1 induces neural differentiation, with increased tendencytowards neuronal over glial lineage.

The pathological manifestation of stroke, at least in this MCAo model,is characterized by extensive neuronal loss accompanied by increasedglial cell activation. Thus, it appears that the neuronal replacementprovided by the Composition 1 supplement is more beneficial than theglial cell replenishment occurring in the control group, given thevehicle-only treatment. The robust neuronal differentiation at two weekspost-stroke is equally advantageous since a rapid cell death cascadeproceeds after the stroke onset. The preferential neuronaldifferentiation during the acute stroke phase provides a solid evidencethat neurogenesis plays a major active role in the Composition 1mediated neuro-protection. Thus, it is believed that there are multipleadvantages to using supplementation effective in repairing damage causedby injuries such as stroke in mammals.

Many other combinations and doses will be apparent to an artisan basedon the examples and ranges provided for combinations of ingredients.Some synergistic effects are shown for combinations of two or more ofthe listed active ingredients. In a composition preferred for protectiveeffect from injury and proliferation and differentiation of stem cells,as shown by in vivo and in vitro results, three or more substances arecompounded in effective, synergistic ratios of blueberry, carnosine,green tea extract and Vitamin D3, with or without equivalent amounts ofVitaBlue, catechins or other substances. Ranges of effective doses maybe tailored for a specific mammal by comparing the amount of eachsubstance measured in blood serum levels, as compared to a known animalor human.

1-9. (canceled)
 10. A composition for stimulation of stem cellproliferation comprising blueberry extract, carnosine, and green teaextract.
 11. (canceled)
 12. The composition of claim 10, wherein thecomposition comprises blueberry extract, carnosine, green tea extractand vitamin D₃.
 13. The composition of claim 10, wherein the compositioncomprises at least 400 milligrams of blueberry extract.
 14. Thecomposition of claim 10, wherein the composition includes green teaextract in a range from 400 mg to 5 grams. 15-29. (canceled)
 30. Acomposition for increasing stem cell proliferation comprising aneffective amount of blueberry extract and carnosine, such that thecombination of at least two substances exerts a synergistic effect onthe stem cell proliferation. 31-36. (canceled)
 37. The composition ofclaim 30 wherein the composition comprises 400 mg to 25 grams ofblueberry extract.
 38. The composition of claim 30 wherein thecomposition contains about 10 mg to about 100 mg carnosine.
 39. Thecomposition of claim 30 wherein the composition comprises 400 mg to 25grams of blueberry extract and about 10 mg to about 100 mg carnosine.40. The composition of claim 10, wherein the composition contains about10 mg to about 100 mg carnosine.
 41. The composition of claim 10,wherein the composition contains about 10 mg to about 100 mg carnosineand 400 mg to 5 grams of green tea extract.
 42. The composition of claim10, wherein the composition contains 400 mg to 25 grams of blueberryextract and 400 mg to 5 grams of green tea extract.
 43. The compositionof claim 10, wherein the composition contains 400 mg to 25 grams ofblueberry extract, about 10 mg to about 100 mg carnosine, and about 400mg to 5 grams of green tea extract.
 44. A method for increasing stemcell proliferation comprising administering a composition comprisingblueberry extract, carnosine, and green tea extract to a human.
 45. Themethod of claim 44, wherein the composition the composition contains 400mg to 25 grams of blueberry extract, about 10 mg to about 100 mgcarnosine, and about 400 mg to 5 grams of green tea extract.
 46. Amethod for increasing stem cell proliferation comprising administering acomposition comprising blueberry extract and carnosine to a human. 47.The method of claim 46, wherein the composition comprises 400 mg to 25grams of blueberry extract and about 10 mg to about 100 mg carnosine.